Background/Purpose: The premise of this study is that the impact of renal injury in lupus nephritis is widespread with consequences to resident cells in other tissue beds, such as non-sun exposed non-lesional skin. Reflection of a relevant pathway in renal tissue by a more readily accessible compartment would be an advance. Single-cell transcriptional states may provide a framework for understanding how in vivo biological function emerges from complex cell ensembles.

Methods: As an approved nested study within the SLE Accelerated Medicines Partnership (AMP), single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of non-lesional non-sun exposed skin from the buttocks of 3 SLE patients with proteinuria and known ISN/RPS Class and 3 healthy controls. Libraries were prepared on the Fluidigm C1 platform followed by sequencing on an Illumina HiSeq 2500. Differential expression was used to assign in vivo cell-type compositions through unsupervised sampling and modeling of transcriptional states in single cells. We generated 36 single-cell data sets with on average 852 genes/cell and 37% of reads mapping to the reference genome. Data are expressed as log2 transcripts per million.

Results: Based on expression of COL1A1, COL1A2, COL3A1, MFAP5 and MFAP4, assignments yielded 12 fibroblasts from 3 patients. From the one Class II subject there were 5 single cell transcriptomes. The other 2 subjects (1 Class IV,V; 1 Class III,V) yielded 7 single cell transcriptomes. From the 3 controls, there were 22 transcriptomes. In the aggregate data for controls, the top one thousand genes were used for a cluster analysis of categories in the DAVID annotation. In the controls, a prominent cluster with an enrichment score of 5.60, in the extracellular matrix category, was significantly represented in the set (P=3.77E-12). The DAVID annotation of skin transcriptomes from subjects with proliferative nephritis resembled the healthy controls; however, closer scrutiny showed differences as highlighted in Table 1 which focuses on the expression of an annotated gene set of  transcripts in the extracellular matrix category (11 are shown) comparing case vs control. In dermal fibroblasts from subjects with proliferative disease the expression of anti-fibrotic genes was increased while pro-fibrotic genes were attenuated. This result suggests that an event involving localized fibrosis of renal tissue yields at distant sites (such as the skin), a fibroblast transcriptome which exerts a countermeasure to forestall fibrosis. The profile of transcripts of subjects with class II resembled controls.

Conclusion: Single-cell RNAseq is feasible and informative in cell specific transcriptome analysis of fresh non-lesional skin biopsies from SLE patients with a spectrum of active renal disease. The expression of fibroblasts and genes reflective of anti-fibrotic pathways support application of this novel approach to study readily accessible tissue.